Retinoic Acid Receptor γ: Specific Immunodetection and Phosphorylation

Synthetic peptides corresponding to cDNA-deduced amino acid sequences unique to the human and mouse retinoic acid receptor γ1 (hRAR-γ1 and mRAR-γ1, respectively) were used to generate anti-RAR-γ1 antibodies. Four mAbs were selected, which were directed against peptides found in region A1 (Ab1γ(A1)), region F (Ab2γ(mF) and Ab4γ(hF)) and region D2 (Ab5γ(D2)). These antibodies specifically immunoprecipitated and recognized by Western blotting RAR-γ1 proteins in COS-1 cells transfected with expression vectors containing the RAR-γ1 cDNAs. They all reacted with both human and mouse RAR-γ1 proteins, except Ab4γ(hF) that was specific for hRAR-γ1. Rabbit polyclonal antibodies, directed against a peptide from the mRAR-γ1 F region were also obtained (RPγ(mF)) and found to be specific for mouse RAR-γ1 protein. Furthermore, in gel retardation/shift assays the antibodies specifically retarded the migration of complexes obtained with a RA response element (RARE). Antibodies raised against regions D2 and F also recognized the RAR-γ2 isoform which differs from RAR-γ1 only in the A region. On the other hand, antibodies directed against the A1 region of RAR-γ1 (Ab1γ(A1)) only reacted with the RAR-γ1 protein. The antibodies characterized here allowed us to detect the presence of mRAR-γ1 and γ2 isoforms in mouse embryos and F9 embryonal carcinoma cells nuclear extracts. They were also used to demonstrate that the mRAR-γ1 protein can be phosphorylated and that the phosphorylation occurs mainly in the NH2-terminal A/B region.